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ISSN: 1314-6246

Vrancheva et al. RESEARCH ARTICLE

J. BioSci. Biotech. 2012, 1(3): 255-259.

Radka Vrancheva 1 Ivan Ivanov 2,3 Andrey Marchev 2 Atanas Pavlov 2,3
Authors addresses: 1 Department of Analytical Chemistry, University of Food Technologies, 4000 Plovdiv, Bulgaria. 2 Laboratory of Applied Biotechnologies, Institute of Microbiology Stephan Angeloff, Bulgarian Academy of Sciences, 4000 Plovdiv, Bulgaria. 3 Department of Organic Chemistry and Microbiology, University of Food Technologies, 4000 Plovdiv, Bulgaria. Correspondence: Ivan G. Ivanov Laboratory of Applied Biotechnologies, Institute of Microbiology Stephan Angeloff, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria. Tel.: +359 32 642430 e-mail: ivanov_ivan.1979@yahoo.com Article info: Received: 25 October 2012 Accepted: 21 December 2012

Qualitative and quantitative determination of protopine in Fumaria spp. by TLCdensitometry method


ABSTRACT
A rapid and accurate TLC-densitometry method for qualitative and quantitative determination of protopine has been developed. The best separation was achieved using a mobile phase chloroform ethyl acetate methanol ammonium hydroxide (80:80:40:0.05, v/v/v/v). The results obtained by this method (CV% 3.4) corresponded well with those obtained by using a HPLC method. The reliability of the proposed method was proved through reproducibility test with alkaloid extracts from Fumaria spp

Key words: TLC, protopine, HPLC, Fumaria officinalis L., Fumaria rostellata Knaf.

Introduction
Plants of the genus Fumaria have been used in the traditional medicine as anti-hypertensive, diuretics, hepatoprotectants and laxatives (to treat gastrointestinal disorders), as well as in the treatment of some skin diseases (rashes or conjunctivitis) (Suau et al., 2002a, 2002b). The biological activity of Fumaria spp. is mostly associated with the presence of isoquinoline alkaloids. The most important isoquinoline alkaloid is protopine (7-methyl-6,8,9,16tetrahydrobis[1,3]benzodioxolo [4,5-c:5',6'-g]azecin-15(7H)one). This alkaloid possess strong hepatoprotective activity (Rathi et al., 2008), inhibits histamine H1 receptors and platelet aggregation (Saeed et al., 1997), inhibits serotonin transporter and noradrenaline transporter and has an antidepressant effect (Xu et al., 2006), as well as antimicrobial, antiviral (Orhana et al., 2007) and anti-

inflammatory activities (Saeed et al., 1997). Several techniques have been used for its determination in plant extracts, including TLC (Tanahashi & Zenk, 1985; Hadjiakhoondi et al. 1999; Waksmundzka-Hajnos & Petruczynik, 2008), GC-MS (Suau et al., 2002a, 2002b; Maiza-Benabdesselam et al., 2007a, 2007b), HPLC (Souek et al., 1999). But until now in the scientific literature has not been described TLC-densitometry method for quantitative determination of protopine from crude alkaloid extracts from Fumaria spp. TLC-densitometry is a suitable method for screening programs of huge number of samples selection of high-producing plant individuals because it is simple, faster and cheaper than HPLC and has appropriate sensitivity and reliability (Berkov & Pavlov, 2004). This study describes a rapid TLC method for quantitative and qualitative determination of isoquiloline alkaloid protopine in total alkaloid extracts from different Fumaria species.

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255

ISSN: 1314-6246

Vrancheva et al. RESEARCH ARTICLE

J. BioSci. Biotech. 2012, 1(3): 255-259.

Materials and Methods


Chemicals and plant material Protopine (95%) was obtained from Extrasynthese (France). Aerial parts of Fumaria officinalis L. were collected from Sozopol and those of Fumaria rostellata Knaf. from Bucino village, near to Blagoevgrad, both in May 2012. Each sample was a mixture of material from several plants. Extraction of alkaloids Intact plants were dried at room temperature and powdered. Samples of 150 mg of drug were moistened with ethanol (3x5ml) in an ultrasonic bath for 15 min. The combined extracts were concentrated under vacuum and dissolved in 22 mL of 3% sulfuric acid. The neutral compounds were removed by extraction (three times) with diethyl ether. The alkaloids were fractionated after basification of the extracts with 1 mL of 25% ammonia and extraction with chloroform (33 mL). The chloroform extracts were then dried over anhydrous sodium sulfate and evaporated to dryness. TLC-analysis A standard solution containing 1 mg/mL of protopine was prepared in ethanol. Aliquots of the stock solution containing 5, 10, 15, 20, 25 and 30 g of standard were spotted together with 15 L of the samples of unknown alkaloid concentration onto silica gel aluminium plates (ALUGRAM SIL G, 20x20), (Macherey-Nagel, Germany) using different solvent systems: 1) Ethyl acetate:methanol:ammonium hydroxide (170:20:10, v/v/v) (Hadjiakhoondi et al., 1999); 2) Petroleum ether:diethyl ether:methanol (100:100:6, v/v/v) (Chelombiko et al., 1971); 3) Chloroform:methanol:ammonium hydroxide (190:10:1, v/v/v) (Tanahashi & Zenk, 1985); 4) Chloroform:ethyl acetate:methanol (80:80:40, v/v/v) (Nino et al., 2006); 5) Chloroform:ethyl acetate:methanol:ammonium hydroxide (80:80:40:0.05, v/v/v/v). The alkaloids were visualized by triplicate spraying with Dragendorff reagent. Ten minutes after the final spraying, the plates were scanned at an optical resolution of 600 dpi using an HP Scanjet 2200c. For the quantification of protopine was used QuantiScan (version 2.1 Biosoft, Cambridge, UK) image analysis software. The protopine content of each sample of unknown alkaloid concentration was calculated from the peak areas of the densitogram by comparison with standard analyzed on the same plate.

HPLC analysis Protopine concentration was defined using Waters 1525 Binary Pump HPLC systems (Waters, Milford, MA, USA), Waters 2484 dual Absorbance Detector (Waters, Milford, MA, USA), equipped with Supelco Discovery HS C18 column (5 m, 25 cm 4.6 mm) and Breeze 3.30 software. Solvents for the preparation of the mobile phases were: I) 10.1 mL of triethylamine added to 1 L redistilled water, adjusted with H3PO4 to pH 2.5 and II) acetonitrile. The mobile phases used were: A: 80% of I and 20% of II (v/v), and B: 40% of I and 60% of II (v/v). The mobile phase program was: 02 min100% A, 215 min 77% A and 23% B, 1530 min 60% A and 40% B (Souek et al., 1999). UV detector was set at 290 nm and the volume of injection was 20 l. For analyses, protopine standard and the alkaloid fractions were dissolved in 1 mL 1 N HCl in ethanol and were injected into the HPLC system. All measurements were performed at 26C and the mobile phase flowrate was 1.0 mL/min. Each determination was repeated five times.

Results and Discussion


The aim of this work was to establish a TLC method for fast screening and selection of protopine-rich plants of the genus Fumaria. In our study we used a different mobile system. When were used the mobile phases 1or 3, there was no sufficient separation and all alkaloids were in one spot at the top of the densitograms (Figure 1). When were used the solvent systems 2 or 4, the alkaloids were at the start line of the TLC-plate. The best results were obtained using following solvent system: chloroform:ethyl acetate: methanol:ammonium hydroxide (80:80:40:0.05, v/v/v/v), (Figure 2). In this case, 7 different alkaloids were separated with Rf values of 0.14, 0.21, 0.28, 0.36, 0.45, 0.80, 0.88 (Figure 2). To improve the spot shapes and separation of isoquinloline alkaloids we basified mobile phase by adding an ammonium hydroxide in small concentration, because most of the them are strong bases. Priority of the developed TLC method was to perform separation of protopine from the mixtures of other accompanying isoquinloline alkaloids from Fumaria spp. As a reference method for the quantification of protopine in this study was used HPLC. The calibration curve was linear in the concentration range 10100 g/mL with correlation coefficient 0.998. Reproducibility of the proposed TLC method is presented in Table 1. Deviations for Fumaria officinalis L. and Fumaria rostellata Knaf. are 3.43 and 3.42.

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ISSN: 1314-6246

Vrancheva et al. RESEARCH ARTICLE

J. BioSci. Biotech. 2012, 1(3): 255-259.

Figure 1. Densitogram obtained from the alkaloid extracted from Fumaria officinalis L. on silica 60 F 254 with: A solvent system 1 ethyl acetate:methanol:ammonium hydroxide (170:20:10, v/v/v); B solvent system 3 chloroform:methanol: ammonium hydroxide (190:10:1, v/v/v).

Figure 2. Densitogram from chromatogram of the alkaloid extracted from Fumaria officinalis L (A) and the protopine (B) on silica 60 F254 with solvent system 5 chloroform:ethyl acetate:methanol:ammonium hydroxide (80:80:40:0.05, v/v/v/v).

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ISSN: 1314-6246

Vrancheva et al. RESEARCH ARTICLE

J. BioSci. Biotech. 2012, 1(3): 255-259.

Table 1. Reproducibility test: determination of protopine in sample F. officinalis L. and F. rostellata Knaf. by TLC and HPLC methods.
Number 1 2 3 4 5 Mean SD CV% Fumaria officinalis L. TLC HPLC 269.6* 262.1 274.6 273.7 282.6 285.4 261.2 268.1 260.7 276.3 269.8 273.1 9.2 8.7 3.4 3.21 Fumaria rostellata Knaf. TLC HPLC 324.9 341.6 316.7 310.8 312.7 330.7 313.7 335.0 339.1 354.3 321.4 334.5 11.0 7.1 3.42 2.13

Legend: *g protopine/g DW

These results corresponded well with those obtained by HPLC (3.21 and 2.13). The difference in qualitative determination of protopine between HPLC and TLC analysis is just 9.70.1% and consequently the TLC-method that has been developed was the reliability for fast screening of protopine and other isoquinloline alkaloids. The distinctions that were observed could be described to the variable conditions of spraying and to differences between the layers themselves. The spraying of the layers is of fundamental importance in obtaining reproducible results (Berkov & Pavlov, 2004). The described TLC method is faster, cheaper and easer for the determination of protopine in comparison with HPLC. Moreover, this method can be applied not only in the laboratory, but also under field conditions for the rapid screening huge number plant individuals of wild populations of Fumaria spp.

Acknowledgement
The work was supported by the Bulgarian Science Foundation, Bulgarian Ministry of Education, Youth and Science (contract DMU 03/77-2011).

References
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